Field of the Invention
The present invention relates to recombinant adenoviruses to regulate angiogenesis and pharmaceutical compositions for preventing or treating angiogenesis-related diseases.
Description of the Related Art
Vascular permeability factor (VPE) was first discovered by Senger group in the year of 19831 and the functions or actions of vascular endothelial growth factor (VEGF) were elucidated in 1989. Thereafter, VPE and VEGF were addressed as the same biomolecule and highlighted due to its critical role in angiogenesis of tumors.
VEGF-A is a potent inducer of angiogenesis and the lack of its single allele causes the inhibition of cardiovascular development to result in lethality in embryos2, which demonstrates the requirement for the elaborate regulation of VEGF-A expression. To date, it has been reported that there are seven human VEGF-A isoforms (VEGF121, VEGF145, VEGF148, VEGF165, VEGF183, VEGF189 and VEGF206), which are generated by alternative splicing of primary VEGF-A mRNA consisting of eight exons3,4. Recent research results suggest that VEGF is also involved in apoptosis inhibition, lymphangiogenesis, immune suppression and hematopoietic stem cell survival5-8.
VEGE-mediated angiogenesis refers to successive processes to induce the growth of new capillary blood vessels from pre-existing blood vessels, and plays a critical role in tumor growth and invasion. VEGF-A is observed to be overexpressed in a variety of tumor cells9. Because a blood supply through angiogenesis is necessary in tumor growth, the inhibition of angiogenesis in tumor becomes an attractive target for anti-cancer therapy and some drugs such as angiostatin, endostatin, thrombospondin-1 and uPA-fragment have been developed as angiogenesis inhibitors10. The high expression of these angiogenesis inhibitors in tumor cells may be a potent therapeutic approach. It has been reported that some angiogenesis inhibitors such as angiostatin exhibit anti-cancer effects in a preclinical trial once they are administered11.
However, angiogenesis inhibitors requires repeated administration due to their shorter half-life, causing serious problems such as toxicity, high cost and difficulty in determining suitable dosage. In contrast, gene therapy has plausible advantages such as high expression of angiogenesis inhibitors by single administration, cost effectiveness and introduction of at least one gene into body12.
Adenoviruses are predominantly used as gene carriers for gene therapy for cancer, having some advantages including high gene transmission efficiency, feasible production at higher titer and convenient concentration. The single administration of replication incompetent adenoviruses expressing angiostatin has been suggested to inhibit the tumor growth by about 80% in U87MG glioma xenograft models13. In this regard, the high expression of anti-angiogenesis agents carried in adenoviruses would give rise to potent therapeutic effect on cancers.
Unlike anti-cancer therapy, therapeutic angiogenesis is a progressive approach aimed at increasing the number of collateral vessels delivering oxygen blood to ischemic tissue14. Conventional therapeutics including β-blockers, Ca2+-antagonists, nitrates [coronary artery disease (CAD) treatment] and prostanoids [peripheral arterial occlusive disease (PAOD) treatment] promote no the growth of collateral vessels10.
Growth factors are demanded to be expressed in initial phase for forming new blood vessels in ischemic tissues. The direct administration of recombinant growth factors is generally considered less inefficient than gene therapy. For example, the administration of VEGF and fibroblast growth factor (FGF) proteins to CAD patients has been shown to exhibit little or no therapeutic effects in clinical trials16,17. Unlikely, recombinant replication-incompetent adenoviruses expressing FGF-3 under the control of CMV (cytomegalo virus) promoter have been reported to be infected to about 25-30% cardiac muscles and induce the growth of new collateral vessels upon administering to coronary artery of pigs. In addition, the intracoronary gene-transfer using adenoviruses increases blood flow and contractile function in an ischemic region of the heart18.
Because blood vessels are easily manipulated for gene transfer and short-term expression of transformed gene is enough to show therapeutic effects on ischemic diseases19,20, an adenoviral gene therapy for ischemic diseases becomes promising. Where even a portion of cardiomyocytes or muscle cells is transformed with a therapeutic gene by gene therapy for ischemic diseases, they could express continuously growth factors for angiogenesis. In addition, since growth factors are required at an initial step for angiogenesis, therapeutic effects would be fully exhibited10. A local long-term expression of VEGF is likely to induce excessive angiogenesis and hemangioma21. The main targets of gene therapy for cardiovascular diseases include promotion of angiogenesis for coronary artery disease (CAD) and peripheral arterial occlusive disease (PAOD) and inhibition of postangioplasty stent restenosis23,24.
To elevate the clinical applicability of gene therapy, it is critical to provide a system for specifically regulating the expression of therapeutic genes. The transcription of a gene is regulated in vivo by transcription factors composed of two functional domains. Of them, a DNA binding domain recognizes and binds to a specific DNA sequence and a regulation domain controls transcription of a gene25. Cys2-His2 zinc finger proteins form the largest family of eukaryotic transcription factors and comprise more than half of transcription factors present in a human genome26. In this regard, zinc finger proteins could provide a pivotal structural platform for preparing artificial transcription factors27.
Zinc finger domains capable of binding to a specific DNA sequence are linked to natural-occurring transcription activation domain or repression domain to provide artificial transcription factors that could recognize target genes and promote or repress their expression. This approach permits to regulate gene expression with no modification of target DNA sequences. Several reports describe that artificial zinc finger proteins could successfully regulate endogenous chromosomal genes28-30.
Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.